Tiwari, M. K. | Search bioRxiv

bioRxiv2019

Dissection of Catalytic Site in Crucial Gut Microbiome Enzyme: Bile Salt Hydrolase

Bile Salt Hydrolases (BSHs) are enzymes from enteric bacteria that catalyze the hydrolysis of Bile Acids and consequently promote the reduction of cholesterol level in the mammalian body. Out of several reported BSHs, the Enterococcus faecalis BSH (EfBSH) has been reported to have the highest enzymatic activity. Herein, we have investigated the mechanistic details of the EfBSH activity. The study was carried out employing two mutants of EfBSH: E269A and R207A, which shows differential catalytic activity. The mutant E269A exhibits significant loss in the BSH activity with an increased affinity towards the substrate as compared to R207A mutant. Further, R207A was found to be involved in allostery with an increased EfBSH activity towards tauro-conjugated bile acids. The structural and electrostatic force analyses of the active sites of the E269A mutant and the wild type EfBSH (wt EfBSH) revealed that the interaction between Glu21 and Arg207 is the determining factor in maintaining the dynamic allostery and high activity of EfBSH.

biochemistry↗

bioRxiv2019

Detection and characterization of a novel copper-dependent intermediate in a lytic polysaccharide monooxygenase

Lytic polysaccharide monooxygenases (LPMOs) are copper-containing enzymes capable of oxidizing crystalline cellulose and the enzyme has large practical application in the process of refining biomass. The LPMO catalytic mechanism still remains debated despite several proposed reaction mechanisms. Here, we report a long-lived intermediate (t[1/2] = 6 - 8 minutes) observed in an LPMO from Thermoascus aurantiacus (TaLPMO9A). The intermediate with a strong absorption around 420 nm is formed when reduced LPMO-Cu(I) reacts with H2O2. UV-vis absorption spectroscopy, electron paramagnetic resonance (EPR), and stopped-flow spectroscopy indicate that the observed long-lived intermediate involves the copper center and a nearby tyrosine (Tyr175). We propose that the reaction with H2O2 first forms a highly reactive short-lived Cu(III)-intermediate which is subsequently transformed into the observed long-lived copper-dependent intermediate. Since sub-equimolar amount of H2O2 to LPMO boosts oxidation of phosphoric acid swollen cellulose (PASC) suggests that the long-lived copper-dependent intermediate is part of the catalytic mechanism for LPMOs. The proposed mechanism offers new perspectives in the oxidative reaction mechanism of copper enzymes and hence for the biomass oxidation and the reactivity of copper in biological systems.

biochemistry↗